Modified peptides and proteins are valuable biophysical tools for studying biological processes, both in vitro and in vivo. They are also useful in assays to identify new drugs and therapeutic agents. In particular, quantitative live cell imaging using fluorescent proteins and peptides is revolutionizing the study of cell biology. An exciting recent development within this field has been the construction of peptide and protein biosensors exhibiting altered fluorescence properties in response to changes in their environment, oligomeric state, conformation upon ligand binding, structure, or direct ligand binding. Appropriately labeled fluorescent biomolecules allow spatial and temporal detection of biochemical reactions inside living cells. See for example Giuliano, K. A., et al., Annu. Rev. Biophys. Biomol. Struct. 1995, 24:405–434; Day, R. N., Mol. Endocrinol. 1998, 12:1410–9; Adams, S. R., et al., Nature 1991, 349:694; Miyawaski, A., et al., Nature 1997, 388:882–7; Hahn, K., et al., Nature 1992, 359:736; Hahn, K. M., et al., J. Biol. Chem. 1990, 265:20335; and Richieri, G. V., et al., Mol. Cell. Biochem. 1999, 192:87–94.
Procedures for site-specific modification of polypeptides have been described, including: chemically selective labeling in solution (Brinkley, M. Bioconjugate Chemistry 1992, 3:2–13) and on resin bound peptides (Hackeng, T., et al., J. Biol. Chem. Submitted); introduction of ketone amino acids through synthetic procedures (Rose, K. J. Am. Chem. Soc. 1994, 116:30–33; King, T. P., et al., Biochemistry, 1986, 25:5774–5779; Rose, K., et al., Bioconjugate Chem. 1996, 7:552–556; Marcaurelle, L. A., Bertozzi, C. R. Tett. Lett. 1998, 39:7279–7282; and Wahl, F., Mutter, M. Tett. Lett. 1996, 37:6861–6864); and molecular biology techniques (Cornish, V. W., et al., J. Am. Chem. Soc. 1996, 118:8150). For example, green fluorescence protein (GFP) is a fluorescent protein that has been fused to other proteins using molecular biology techniques and has been used to visualize intracellular proteins (see, e.g., Katz et al., BioTechniques 25: 298–304 (1998)).
While each of these methods has utility for producing a particular class of biosensor or labeled polypeptide, all have limitations that restrict their general use. Labeling of natural amino acid side-chains in solution is often impractical because of the existence of many other competing nucleophiles. Additionally, the use of unnatural amino acids, such as those bearing ketones for selective labeling, requires the synthesis of dye constructs or amino acids that are difficult to make and are not available commercially. While a variety of proteins have been fused to GFP, some GFP-labeled proteins fail to fluoresce. Mutational analysis indicates that the structure of GFP is extremely sensitive to molecular or biochemical modifications. Moreover, GFP is extremely large (238 amino acids). Hence, fusion of GFP to other proteins can alter the function of GFP or even the function of the protein to which it is fused. Moreover, GFP does not have the diverse capabilities of smaller, synthetic molecules, some of which provide a variety of fluorescence wavelengths, or are capable of reporting on protein conformation, or can photo-crosslink or act as NME or EPR probes. Accordingly, new fluorescent molecules with more diverse properties and new attachment methods are needed.
Currently, the major obstacles to the development of fluorescent biosensors and labeled polypeptides remain: (1) The difficulty in site-specific placement of the dye in the polypeptide and (2) determining exactly which site is optimal for dye placement (Giuliano, K. A., et al., Annu. Rev. Biophys. Biomol. Struct. 1995, 24:405–434). Solvent-sensitive dyes and other biophysical probes must be placed precisely for optimal response to changes in protein structure without interference with biological activity. Also, the need for site-specific incorporation of two dyes without impairment of biological activity has proven a serious limitation for utilization of fluorescence resonance energy transfer (FRET) within a single protein. Total chemical synthesis of proteins provides a potential solution to these problems (Wilken, J., Kent, S. B. H. Curr. Op. Biotechnology. 1998, 9:412; Kent, S. B. H. Ann. Rev. Biochem. 1988, 57, 957–989; Dawson, P. E., et al., Science 1994, 266:776–779; Muir, T. W., et al., Proc. Natl. Acad. Sci. 1998, 95:6705–6710; and Cotton, G. J., et al., J. Am. Chem. Soc. 1999, 121:1100–1101). However, many biophysical probes suitable for fluorescent biosensors or other purposes are not stable to the various conditions used for peptide synthesis, and site-specific incorporation after synthesis has been difficult to achieve.
Moreover, labeling with hydrophobic dyes such as thionine or methylene blue can be problematic because these dyes autoaggregate in aqueous solution at high concentration. See, J. Am. Chem. Soc. 63, 69 (1941). These aggregates cause a change in the absorption spectrum and a reduction in the fluorescence of the dyes. Cyanines and merocyanines are also thought to aggregate, causing a quenching of fluorescence (J. Phys. Chem. 69, 1894 (1965)). Such aggregation interferes with conjugation of these fluorescent dyes to other molecules such as proteins. Moreover aggregation by cyanines and merocyanines can be exacerbated after the dyes are conjugated. For example, Waggoner et al. have observed an aggregation phenomenon following the conjugation of cyanin isothiocyanate with an antibody (Cytometry 10, 11–19 (1989)). Fluorescence of a conjugate between a cyanin fluorescent dye and an anti-HCG antibody (molar ratio=1.7) named CY5.18 is quenched in comparison with that of the free cyanin (see U.S. Pat. No. 5,268,486 and Anal. Biochem. 217, 197–204 (1994)). Also, while cyanines are generally stable, inexpensive, simple to conjugate to other molecules and of a suitable size for the recognition of small molecules, they do not change their fluorescence in response to environmental factors, such as solvent polarity. New dyes that eliminate these problems are needed.
Thus, there is currently a need for new fluorescent dyes and peptide synthons having protected functional groups that can be selectively modified to incorporate one or more functional molecules (e.g. a fluorescent label) following peptide synthesis. There is also a need for proteins and antibodies with biophysical probes attached to precise locations, and for simple, non-destructive methods of making such labeled proteins and antibodies. Simpler methods for using these labeled peptides, proteins and antibodies in vivo as biosensors are also needed.